当归多糖改善大鼠心肌缺血再灌注损伤的体内外观察及其机制探讨  

Angelica sinensis polysaccharide ameliorates MIRI in rats in vitro and its mechanism

作　　者：叶建华[1] 沈盛晖[1] 张田杰[1] 冯频频 YE Jianhua;SHEN Shenghui;ZHANG Tianjie;FENG Pinpin(Department of Cardiology,Tongde Hospital of Zhejiang Province,Hangzhou 310012,China;不详)

机构地区：[1]浙江省立同德医院心血管科,杭州310012 [2]浙江省立同德医院药剂科

出　　处：《山东医药》2023年第29期45-50,共6页Shandong Medical Journal

基　　金：浙江省中医药科技计划科研基金项目(2021ZA032)。

摘　　要：目的建立大鼠心肌缺血再灌注损伤(MIRI)的体内、体外模型,观察当归多糖(ASP)对大鼠MIRI的改善作用并探讨其机制。方法在体内研究,成年SD大鼠随机分为假手术组、MIRI组、ASP低剂量组、ASP高剂量组,除假手术组外,均采用冠状动脉左前降支结扎再灌注的方法建立MIRI模型,ASP低、高剂量组在建模后分别给予50、100 mg/kg的ASP灌胃,假手术组及MIRI组给予等量生理盐水灌胃,持续4周;采用ELISA法检测各组血清心肌损伤标志物肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)及炎症因子肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β;Western blotting法检测各组心肌组织凋亡蛋白B淋巴细胞瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)、裂解的半胱胺酸蛋白酶(cleaved Caspase-3)及toll样受体4(TLR4)/核因子κB(NF-κB)通路相关蛋白TLR4、磷酸化核蛋白(p-p65)、磷酸化NF-κB抑制蛋白α(p-IκBα)表达。在体外研究,将大鼠心肌细胞分为对照组、H/R组、H/R+ASP低剂量组、H/R+ASP高剂量组,对照组正常培养不做处理,其他3组均建立体外心肌细胞缺氧复氧(H/R)模型,建模后H/R+ASP低、高剂量组分别加入浓度为5、10µg/mL的ASP,H/R组不做处理;采用MTT比色法检测各组心肌细胞活性,ELI-SA法检测各组心肌细胞炎症因子TNF-α、IL-6、IL-1β,Western blotting法检测各组心肌细胞凋亡蛋白Bax、Bcl-2、cleaved Caspase-3及TLR4/NF-κB通路蛋白TLR4、p-p65、p-IκBα表达。结果在体内研究,血清心肌损伤标志物LDH、CK-MB水平及血清炎症因子TNF-α、IL-6、IL-1β水平MIRI组>ASP低剂量组>ASP高剂量组>假手术组(P均<0.05);心肌组织Bax、cleaved Caspase-3蛋白表达MIRI组>ASP低剂量组>ASP高剂量组>假手术组,心肌组织Bcl-2蛋白表达假手术组>ASP高剂量组>ASP低剂量组>MIRI组(P均<0.05);心肌组织TLR4/NF-κB通路相关蛋白TLR4、p-p65、p-IκBα表达MIRI组>ASP低剂量组>ASP高剂量组>假手术组(P均<0.05)。在体外研究,�Objective To establish in vivo and in vitro models of myocardial ischemia-reperfusion injury(MIRI)in rats,to observe the improvement effect of Angelica polysaccharide(ASP)on MIRI rats and to explore its mechanism.Methods In vivo study,adult SD rats were randomly divided into the sham group,MIRI group,low-dose ASP group and high-dose ASP group.Except the sham group,the MIRI models were established by ligation and reperfusion of the left an⁃terior descending branch of coronary artery in the other groups.After modeling,the rats in the low-dose and high-dose ASP groups were given 50 and 100 mg/kg ASP by intragastric administration,once a day,for 4 weeks,respectively.Rats in the sham group and MIRI group were given the equal volume of normal saline.Serum markers of myocardial injury such as creatine kinase-MB(CK-MB),lactate dehydrogenase(LDH),tumor necrosis factor-α(TNF-α),interleukin6(IL-6),and IL-1βwere detected by ELISA.The expression levels of apoptotic proteins Bax,Bcl-2,cleaved Caspase-3,and TLR4/NF-κB pathway-related proteins TLR4,p-p65,and p-IκBαwere detected by Western blotting.In vitro study,H/R cardiomycetes(derived from neonatal cell culture)were divided into the control group,hypoxia reoxygenation(H/R)group,low-dose H/R+ASP group and high-dose H/R+ASP group.The control group was cultured normally without treat⁃ment,and in vitro cardiomyocyte H/R models were established in the other three groups.After modeling,the low-dose and high-dose H/R+ASP groups were added with 5 and 10μg/mL ASP,respectively,and the H/R group was not treated.MTT colorimetric assay was used to detect cardiomyocyte activity,and ELISA was used to detect inflammatory cytokines TNF-α,IL-6 and IL-1β.Western blotting was used to detect apoptotic proteins Bax,Bcl-2,cleaved Caspase-3,and TLR4/NFκB pathway proteins TLR4,p-p65,and p-IκBαin each group.Results In vivo,the levels of serum myocardi⁃al injury markers(LDH and CK-MB)and serum inflammatory factors(TNF-α,IL-6 and IL-1β)ranked from high to low:MIRI group,low-dose ASP

关 键 词：当归多糖 TLR4/NF-κB信号通路 炎症反应 细胞凋亡 心肌缺血再灌注损伤 

分 类 号：R542.2[医药卫生—心血管疾病]